Journal: Nature Communications
Article Title: Human immunoglobulin G hinge regulates agonistic anti-CD40 immunostimulatory and antitumour activities through biophysical flexibility
Figure Lengend Snippet: Divergent hIgG agonism and specific FcγR-binding requirement. a Diagram showing the OVA-specific CD8 + T-cell response model. In brief, FcγR-humanized (hFCGR Tg ) or -deficient (FcγRα −/− ) mice were adoptively transferred with OT-I cells on day-1, immunized intraperitoneally (i.p.) with 2 μg of DEC-OVA in the presence of control or anti-CD40 antibodies on day 0. Splenocytes were harvested to quantify OVA-specific CD8 + T cells on day 6. b , c Representative FACS profile ( b ) and quantification ( c ) showing the percentage of OT-I cells (CD45.1 + TCRVα2 + ) among CD8 + T cells in mice treated and analyzed as in ( a ) together with 30 μg of indicated control or anti-mCD40 antibodies. Numbers of mice: b , c three hFCGR Tg mice for Ctrl IgG, five hFCGR Tg , and three FcγRα −/− mice for other groups. d – f Quantification of OT-I cells as the percentage of OT-I cells among CD8 + T cells ( d , e ) or cell count ( f ) in FcγR-humanized mice treated and analyzed as in ( a ) together with 10 μg ( d , e ) or 30 μg ( f ) of indicated control or anti-mCD40 antibodies (the N297A mutation abrogates Fc–FcγR binding) and with/without FcγRIIB-blocking antibody 2B6 (150 μg per mouse) ( f ). Numbers of mice: d four mice per group; e five mice per group; f two mice for Ctrl IgG, five mice for αmCD40:G2, six mice for αmCD40:G2 + 2B6. g Quantification of OT-I cells as the percentage of OT-I cells among CD8 + T cells in mice of indicated genotypes (FcγR-deficient (FcgRα −/− , five mice per group), FcγRIIB-deficient (Fcgr2b −/− , five mice per group) or humanized (Fcgr2b −/− hFCGR2B Tg , four mice per group)) treated and analyzed as in ( a ) together with 10 μg of indicated control or IgG2 anti-mCD40 antibodies. Each symbol represents an individual mouse. Bars represent the mean ± SEM. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; unpaired two-tailed t test ( c , g ), one-way ANOVA with Holm–Sidak’s post hoc ( d – f ). Source data ( c – g ) are provided as a Source Data file. A representative of two independent experiments is shown
Article Snippet: CD40-binding ELISA and FACS Binding of anti-CD40 antibodies to mouse CD40 was analyzed by ELISA.
Techniques: Binding Assay, Mouse Assay, FACS, Cell Counting, Mutagenesis, Blocking Assay, Two Tailed Test